I. Field of the Invention
The present invention relates generally to the field of cancer diagnosis and treatment, and more specifically relates to the use of laser scanning cytometry for the determination of endothelial cell death, endothelial tumor cell death and tumor blood vessel density. This method can be used as an indicator for therapeutic response and for determining treatment protocols.
II. Description of Related Art
Angiogenesis, or new blood vessel growth, has become a topic of major prominence in both the scientific literature and the popular press over the past several years. Public interest centers on scientific studies and clinical trials now underway that have demonstrated that inhibitors of angiogenesis can substantially reduce or even stop growth of solid tumors. This finding has lead to much interest and investment in angiogenesis research.
A new area for cancer treatment involves the use of anti-angiogenetic drugs, several of which are already in clinical trials. Angiogenesis is the process by which new blood vessels are formed. Although the exact mechanism of angiogenesis activation remains unknown, researchers have identified the existence of diffusable protein factors released by tumor cells that serve as angiogenesis-stimulators. Recent advances in cancer treatment are based on the fact that tumors, like other cells, require oxygen and other nutrients for growth and proliferation. Once the size of a tumor becomes greater than about 2 mm, a new supply of nutrients is needed for continued growth. Tumors may remain small and dormant state for an indefinite period of time. However, once tumors acquire the capacity induce angiogenesis and produce their own blood vessels, they may grow and metastasize to other regions of the body.
Anti-angiogenic therapy involves the inhibition of tumor growth by preventing the formation of new blood vessels. Scientific studies and clinical trials now underway that have demonstrated that inhibitors of angiogenesis can substantially reduce or even stop growth and metastasis of solid tumors. The major advantages of anti-angiogenic therapy is that these drugs, unlike chemotherapy, radiation therapy, and other traditional cancer treatments, have only mild side effects. The therapy does not cause the death of normal, healthy cells. Also, the body does not acquire resistance to treatment with anti-angiogenic compounds as it does with chemotherapeutic compounds.
Several methods for determining the efficacy of anti-angiogenic compounds exist and include the use of TUNEL (terminal deoxynucleotidyltransferase-uridine nick end labeling) and other labeling methods and a manual method for the determination of apoptosis in tumor cells. For example, see Dong et al. (1999), Ikeda et al. (1996), Shabisgh et al. (1999). Fluorescent dual-labeling of apoptotic endothelial cells has been shown by Shaheen et al., (1999). A method of determining the metastatic potential of a tumor by determining the presence of the pro-angiogenic factor VEGF transcripts is disclosed in U.S. Pat. No. 5,942,385.
Tumor vascularization also is used as a diagnostic tool for tumor growth and metastasis (Folkman et al., 1987). Determining vascularity of tumor tissue by immunohistochemical and non-invasive methods have been done by Fanelli et al. (1999), Griffey et al. (1998), Visscher et al. (1993), Seifert et al. (1997), and in U.S. Pat. No. 5,840,507. Other methods of determining vascular parameters include the use of magnetic resonance imaging (MRI) (U.S. Pat. No. 6,009,342) and the use of color doppler signals (Cosgrove et al., 1990). The use of computers to assist in the determination of vascular parameters has been demonstrated in U.S. Pat. Nos. 5,688,694 and 65,616,469 and by Fox et al., (1995) and has aided in the counting procedures. However, counting of microvessels in tumor samples, besides being labor-intensive, is a qualitative art. The method requires considerable technical training to obtain reliable and reproducible results, and inter-investigator variability is a significant problem. Difficulties in reproducing the method have been reported by several groups (Wiedner, 1995). Additionally, the process of preparing specimens for histology and counting vessels is time consuming. Therefore, the application of this technique has been limited generally to research purposes.
For the techniques described above, a waiting period after anti-cancer therapy is usually required before tumor vascularization can be determined. When using non-invasive methods of observing effects of cancer treatments, one must wait several weeks before changes in the tumor can be observed. This can cause problems, especially if practitioners need to know the efficacy of treatments that have substantial adverse side effects.
One concern that has arisen as anti-angiogenic therapies enter clinical trial is that anti-vascular agents may not be specific for the tumor vasculature. Because previous studies of angiogenesis have identified tumor blood vessels by staining them with antibodies to endothelial cell antigens (CD31, CD34), it has been difficult to directly evaluate potential toxicity to adjacent normal tissue.
A purpose of this invention is to determine whether apoptosis can be measured in tumor endothelial cells in situ and to use these methods to measure patient responses to anti-angiogenic and more traditional therapies; in other words, to confirm that the drug target has been hit and that the desired biological effects have been obtained.
Another purpose if this invention is to provide of method of determining anti-cancer therapeutic efficacy that can be used during or immediately after therapy.
While determination of apoptosis and vascular parameters of tissue are known in the art, A single method in which all of these parameters are determined is not available.